Molecular dynamic simulations reveal an exposed heme edge that has not been observed in any crystal or NMR structure of cytochrome b5. This cleft is likely to be of functional importance for recognition of a redox partner for mediating direct electron transfer for some complexes. In order to study dynamics of the cleft breathing we initiated a steady-state fluorescence study of single Trp-22. Acrylamide quenching of the single tryptophan rat cytochrome b5 and its various disufide mutants was examined by ultrafast time-resolved spectroscopy. In order to probe dynamics of the putative cleft, an additional tryptophan has been placed on the surface (S18W). Comparison of lifetime distribution and anisotropy decays of wild-type and mutant with Trp-18 may provide new information about mobility of the protein surface in the cleft region of the protein.